Molecular and Cellular Approaches to Octopus Arm Regeneration and Development

Octopus arm regeneration, following amputation, includes the reconstruction of the massive axial nerve cord, the morphologically complex suckers, and elements of the adaptive coloration system. This regeneration is rapid, scarless, and can proceed across several arms simultaneously. Here, I present the first comprehensive investigation of arm regeneration in octopuses with their full developmental history known. One of the first events of octopus arm regeneration is the rapid innervation of the wound plane by 3 days post-amputation (D3). By D7, a blastema of proliferating cells has accumulated beneath the wound epithelium. This blastema is not a homogeneous structure, instead containing distinct cellular zones, restricted expression of patterning genes and a zone of invading neuronal processes. By D21, the gross tissues of the arm have been restored, though several weeks of growth are needed to reach the length of the contralateral arm. A key finding of this thesis is a previously unknown systemic proliferative response following injury, such as arm or single sucker amputations, or skin wounding. This proliferative response is extremely rapid, detected within 2 hours of injury, and extends across the entire arm crown, including the seven uninjured arms. This long-range response may have important implications for the origin of octopus blastemal cells. This thesis, in defining the cellular and molecular characterization of the early octopus blastema, opens new avenues for understanding the roles of innervation and cell-type differentiation in regeneration.

Octopus arms have muscular, chemotactile suckers critical for locomotion, object manipulation, and prey capture. My study of over 700 Octopus bimaculoides embryos and juvenile octopuses reveals a tight regulation of sucker number and spatial patterning. Initially, three suckers form in a straight line at the proximal base of each arm, next to the beak. Suckers are added in a zigzag pattern, initiated posteriorly, throughout the octopus’s life. Strikingly, the bilateral symmetry of sucker number on each arm pair is maintained through at least six weeks post-hatching, when there are around 50 suckers per arm. In early embryonic stages, sucker numbers are identical across all eight arms. From stage 17 onwards, arms 2 and 3 have the same number of suckers, while arms 1 are shorter in length, with fewer suckers. Arms 4 are more variable but always preserve bilateral symmetry within each octopus. Further insight into the regulatory features of their patterning emerged from an abnormal clutch of 36 hatchlings, all of which displayed irregularities in arm number, length, and sucker arrangement. Our findings highlight an unexpected spatial quantitative control of sucker development in the octopus and establish that each octopus arm pair has a distinct identity well before hatching.